Cells regulate their volume not only to counteract swelling or shrinkage caused by osmotic challenges, but also during processes like cell growth, division, and migration. As water transport across cellular membranes is driven by osmotic gradients, cell volume regulation requires appropriate changes of intracellular concentrations of ions or organic osmolytes like taurine (Hoffmann et al., Pasantes-Morales et al.). Regulatory volume decrease (RVD) follows the extrusion of intracellular Cl− and K+ ions, as well as other osmolytes, across the plasma membrane. A key player is the volume-regulated anion channel (VRAC) that mediates characteristic swelling-activated Cl− currents (ICl(swell)), and appears to be ubiquitously expressed in vertebrate cells (Nilius et al. 1997, Okada et al. 2009, Okada et al. 1997). Nearly inactive under resting conditions, VRAC is slowly opened by hypotonic swelling. The mechanism by which cell swelling leads to VRAC opening remains enigmatic, with a plethora of studies suggesting a confusingly complex and controversial signal transduction cascade.
VRAC currents are outwardly rectifying (hence the alternative name VSOR for volume stimulated outward rectifier (Okada et al. 2009, Okada et al. 1997)) and show variable inactivation at inside-positive voltages. VRAC conducts iodide better than chloride and is inhibited by several rather non-specific compounds. VRAC might also conduct organic osmolytes like taurine (Jackson et al., Mulligan et al., Roy et al.) (hence the name VSOAC, volume-stimulated organic osmolyte/anion channel (Strange et al.)), but this notion has remained controversial (Lambert et al., Shennan et al., Stutzin et al.). VRAC activity is believed to be not only important for cell volume regulation per se, but also for basic cellular functions like the regulation of cell cycle, proliferation and migration (Hoffmann et al., Nilius et al. 1997, Okada et al. 2009). It is thought to play a role in apoptosis and various pathological states including sickle cell anemia, ischemic brain edema and cancer (Okada et al. 1997, Okada et al. 2006), and VRAC-mediated membrane depolarization may trigger swelling-induced exocytosis (Moser et al.). However, progress in the characterization of VRAC and its biological roles has been severely limited by the failure to identify the underlying protein(s) despite intense efforts for more than two decades.
Hence, the identification of modulators, such as inhibitors or activators, of VRAC could enable the development of medically relevant compounds for potential use in the modulation of medical conditions associated with changes in cell volume, such as those mentioned above.
The lack of specific high-affinity inhibitors of ICl(swell) has precluded the biochemical identification of VRAC. Expression cloning approaches have been hampered by the ubiquitous expression pattern of the channel, its complex regulation, and potentially by a heteromeric architecture. Nonetheless, several proteins were suggested to embody VRAC. These proteins include the multidrug-resistance (mdr) P-glycoprotein (Valverde et al.) which is a transport ATPase, plCln, (Paulmichl et al., Fürst et al.) which turned out to be a spliceosome component (Pu et al.), as well as ClC-3 (Duan et al.) which rather is an endosomal anion transporter (Stobrawa et al.). All these claims were disproved by further experimentation (Stobrawa et al., Voets et al., Wine et al., Tominaga et al., Gong et al.). ClC-2 Cl− channels activate upon cell swelling, but their inward rectification and Cl− over I− selectivity deviate from VRAC (Gründer et al.). Drosophila dBest1, a member of a family of bona fide Ca2+-activated Cl− channels (Hartzell et al.), mediates swelling-activated Cl− currents in insect cells (Stotz et al., Chien et al.), but their characteristics differ from VRAC currents and the closest mammalian homolog of dBest1 is swelling-insensitive (Fischmeister et al.).
The present invention is based on a structural and functional characterisation of VRAC and its components. VRAC represents a structurally new class of channel that conducts ions and organic osmolytes. The invention relates to a practical utilisation of the VRAC characterisation shown herein, in order to provide methods and compounds used for identifying VRAC modulators in addition to the VRAC components themselves.